【摘要】： 流感病毒是一種對(duì)人類(lèi)健康和公共衛(wèi)生威脅很大的病毒,每年全球有很多人由于流感病毒感染致病或死亡。其中老年人對(duì)流感病毒的抵抗力尤為低下,更易受到流感病毒感染及其誘發(fā)的并發(fā)癥導(dǎo)致的住院及死亡。因而迫切需要研究能夠特別適合老年人應(yīng)用的安全有效的流感疫苗。闡明免疫衰老的原因,從而啟示和惠及其他老年疫苗的開(kāi)發(fā)和應(yīng)用。 本研究利用昆蟲(chóng)桿狀病毒表達(dá)系統(tǒng)共表達(dá)H1N1流感病毒A/Perto Rico/8/34的HA,NA和M1蛋白,在體外組裝成流感病毒樣顆粒(Virus-like particles, VLPs)。經(jīng)鑒定和純化的VLPs保持了與流感病毒粒子相似的形態(tài)大小以及表面糖蛋白的正常生物學(xué)活性。低劑量VLPs免疫的成年及老年小鼠與滅活流感病毒(Inactivated influenza virus, IIV)相比顯示相同的免疫原性,均能夠保護(hù)被免疫小鼠在致死流感病毒攻擊中存活。但是老年小鼠的保護(hù)效果明顯劣于成年小鼠,表現(xiàn)為老年小鼠體重下降程度大于成年小鼠以及攻毒后體內(nèi)活躍的高水平的抗原特異性CD8 T細(xì)胞反應(yīng)。增大免疫劑量能顯著加強(qiáng)疫苗在老年小鼠中的免疫保護(hù)效果,表現(xiàn)為攻毒中輕微的可以忽略不計(jì)的體重下降以及攻毒后與成年小鼠相當(dāng)?shù)牡退降腃D8 T細(xì)胞反應(yīng)。 雖然流感病毒VLPs能對(duì)被免疫老年小鼠提供完全保護(hù),但是其并不能取得像成年小鼠一樣良好的保護(hù)效果。這樣的結(jié)果激起了我們對(duì)免疫衰老機(jī)制研究的興趣。與其他文獻(xiàn)報(bào)道相似,在實(shí)驗(yàn)中我們發(fā)現(xiàn)老年小鼠體內(nèi)的調(diào)節(jié)性T細(xì)胞(T regulatory cells, Treg)含量顯著高于成年小鼠Treg細(xì)胞約2倍之多。因?yàn)門(mén)reg細(xì)胞對(duì)免疫系統(tǒng)的調(diào)節(jié)性作用,我們推測(cè)這部分累積的Treg細(xì)胞可能是造成老年小鼠接種后疫苗免疫反應(yīng)性低下的原因之一。因此在免疫小鼠前,我們給小鼠腹腔注射CD25的單克隆抗體以暫時(shí)消除Treg細(xì)胞。實(shí)驗(yàn)結(jié)果非常確實(shí)的顯示了消除Treg細(xì)胞能夠極大提高老年小鼠的特異性疫苗免疫反應(yīng),并對(duì)老年小鼠提供完美的免疫保護(hù)力。實(shí)驗(yàn)結(jié)果證明了老年小鼠體內(nèi)積累的Treg細(xì)胞是造成其免疫反應(yīng)低下的主要原因之一。 在闡明了老年小鼠免疫衰老的可能機(jī)制后,我們對(duì)老年小鼠進(jìn)行了一次免疫攻毒保護(hù)試驗(yàn)。以觀(guān)察是否消除Treg細(xì)胞能夠提高疫苗一次免疫的保護(hù)成功率。實(shí)驗(yàn)結(jié)果再一次證實(shí)了Treg細(xì)胞對(duì)調(diào)節(jié)老年小鼠免疫反應(yīng)的重要性:注射過(guò)CD25抗體的免疫老年小鼠在大劑量致死流感病毒攻擊中全部存活且體重鮮有下降,而沒(méi)有注射過(guò)CD25抗體的老年小鼠僅有67%存活,且存活個(gè)體有明顯的發(fā)病癥狀和體重降低。 本研究結(jié)果證明昆蟲(chóng)桿狀病毒生產(chǎn)的流感病毒VLPs在老年小鼠中具有和IIV疫苗相等同的免疫保護(hù)效力。VLPs具有能夠刺激樹(shù)突狀細(xì)胞—專(zhuān)職抗原遞呈細(xì)胞產(chǎn)生細(xì)胞因子的能力。流感病毒VLPs代表了一種新型流感疫苗策略,能夠?qū)Τ赡旰屠夏晷∈筇峁└行У拿庖弑Ｗo(hù)效力,因而具有非常廣闊的應(yīng)用前景。本研究同時(shí)闡明,小鼠免疫衰老的潛在機(jī)制——即Treg細(xì)胞的累積性增多是造成老年小鼠免疫力低下的主要原因之一。免疫接種前暫時(shí)性消除Treg細(xì)胞能夠極大恢復(fù)老年小鼠被抑制的免疫反應(yīng),增強(qiáng)疫苗免疫誘導(dǎo)的體液和細(xì)胞免疫反應(yīng)。針對(duì)Treg細(xì)胞的疫苗研發(fā)策略為今后老年疫苗的研究和開(kāi)發(fā)提供了新思路和可能的靶向及解決方案。
[Abstract]:Influenza virus is a kind of virus that threatens human health and public health. Many people all over the world suffer or die from influenza virus infection every year. Safe and effective influenza vaccines that are especially suitable for the elderly. To clarify the causes of immune aging, and thus inspire and benefit the development and application of other vaccines for the elderly.
In this study, the HA, NA and M1 proteins of H1N1 influenza virus A/Perto Rico/8/34 were co-expressed by insect baculovirus expression system and assembled into influenza virus-like particles (VLPs) in vitro. The VLPs identified and purified maintained the similar morphology and size as influenza virus particles and the normal biological activity of surface glycoproteins. Compared with inactivated influenza virus (IIV), adult and aged mice immunized with low dose of VLPs showed the same immunogenicity and could protect the immunized mice from lethal influenza virus attack. Higher levels of antigen-specific CD8 T cells were active in adult mice and in vivo after challenge. Increasing the dose of the vaccine significantly enhanced the immune protective effect of the vaccine in the aged mice, which showed slight negligible weight loss during attack and low levels of CD8 T cells after challenge.
Although influenza virus VLPs can provide complete protection to immunized aged mice, they can not achieve as good protection as adult mice. This result arouses our interest in the study of the mechanism of immune aging. Because Treg cells regulate the immune system, we speculate that this accumulation of Treg cells may be one of the causes of the decreased immune response in the vaccinated aged mice. Monoclonal antibodies were used to temporarily eliminate Treg cells. The results showed that eliminating Treg cells could greatly improve the specific immune response of the aged mice and provide perfect immune protection for the aged mice. One of the main reasons.
After elucidating the possible mechanism of immune senescence in aged mice, we conducted an immune challenge protection test in aged mice to see if eliminating Treg cells could increase the success rate of vaccine immunization. The immunized mice survived the lethal attack of influenza viruses at high doses with little weight loss, while only 67% of the aged mice without CD25 antibody survived, and the survivors had significant symptoms and weight loss.
The results of this study demonstrated that the influenza virus VLPs produced by insect baculovirus have the same immune protective effect as the IIV vaccine in the aged mice. VLPs have the ability to stimulate the production of cytokines by dendritic cells-specialized antigen-presenting cells. Influenza virus VLPs represent a novel influenza vaccine strategy that can be used for adulthood and development This study also elucidates that the cumulative increase of Treg cells, the underlying mechanism of immune senescence in mice, is one of the major causes of immunodeficiency in older mice. Temporary elimination of Treg cells before vaccination can greatly restore immunity. Inhibited immune responses in aged mice enhance the humoral and cellular immune responses induced by vaccines. Vaccine development strategies for Treg cells provide new ideas and possible targets and solutions for future research and development of vaccines for the aged.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392

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本文編號(hào)：2209081